Published March 15, 1991
Nucleotide Specificity of the Enzymatic and Motile Activities of
Dynein, Kinesin, and Heavy Meromyosin
Takashi Shimizu,* K i y o t a k a Furusawa,* Shinichi Ohashi,* Yoko Y. Toyoshima,* M a k o t o Okuno,§
F a d y Malik,ll a n d R o n a l d D. Valell
*Research Institute for Polymers and Textiles, Higashi, Tsukuba, Ibaraki 305, Japan; *Ochanomizu University, Faculty of Science,
Department of Biology, Ohtsuka, Bunkyo, Tokyo 112, Japan; §University of Tokyo, Faculty of Arts and Sciences, Department of
Biology, Komaba, Merguro, Tokyo 153, Japan; and ItUniversity of California San Francisco, Cell Biology Program,
Department of Pharmacology, San Francisco, California 94143
Abstract. The substrate specificities of dynein, kine-
kinesin and heavy meromyosin, the relative velocities
of filament translocation did not correlate well with
the relative filament-activated substrate turnover rates.
Furthermore, some ATP analogues that did not support the filament translocation exhibited filamentactivated substrate turnover rates. Filament-activated
substrate turnover and power production, therefore,
appear to become uncoupled with certain substrates.
In conclusion, the substrate specificities and coupling
to motility are distinct for different types of molecular
motor proteins. Such nucleotide "fingerprints" of enzymatic activities of motor proteins may prove useful as
a tool for identifying what type of motor is involved
in powering a motility-related event that can be reconstituted in vitro.
UKARYOTIC ceils exhibit many types of motility, including muscular contraction, ciliary or flagellar
movement, chromosome movement, and organelle
transport. Two general classes of motility mechanisms have
been defined according to the "track" on which the "molecular motor" works. Movements along actin filaments are
powered by various myosins, while dynein (Gibbons and
Rowe, 1965; Gibbons, 1988) and kinesin (Vale et al., 1985)
motors power movements along microtubules. All known
myosin move unidirectionally toward the actin barbed end,
and until recently it was thought that dyneins move towards
the microtubule minus end and kinesins move towards the
microtubule plus end. However, a kinesin-like motor (the
product of the ned gene) from Drosophila induces minus
end-directed microtubule movement (Walker et al., 1990).
Other motors may have the capacity to move bidirectionally
along microtubules (Schliwa et al., 1991).
Muscle myosin, ciliary or flagellar dynein and kinesin are
structurally quite different from one another. Muscle myosin
has two globular beads connected to an a-helical coiled-coil
tail (Lowey et al., 1969). Kinesin has somewhat similar
structure to myosin, but is considerably smaller, also having
a tail with fan-shaped tip (Hirokawa et al., 1989). Dynein,
in contrast, has no tails but has two or three heads connected
by stems to a common base (Johnson and Wall, 1983; Johnson, 1985; Toyoshima, 1987). 14S dynein from Tetrahymena
cilia, whose localization in cilium is still controversial, is unlike other other dyneins in having only one head with or
without a short tail (Marchese-Ragona et al., 1988).
Myosin, dynein, and kinesin all turn over the substrate,
ATP, by a rather similar mechanism (Johnson, 1985; Hibberd and Trentham, 1986; Hackney, 1988). The ATP turnover cycle consists of binding to the active site, hydrolysis,
and product release. For those molecular motor enzymes,
the ATP binding and hydrolysis are rapid but the release of
products (ADP and phosphate) is slow and rate limiting: it
is this release of products that is linked to the power
production.
Although the kinetic mechanisms of ATP turnover are
similar for all three mechanochemical enzymes, other characteristics of the ATPase, including the substrate specificity,
are somewhat different. Unlike myosin, dynein has been
shown to have a highly preferred substrate specificity (Gibbons, 1966; Ogawa and Mohri, 1972). Recently we con-
© The Rockefeller University Press, 0021-9525/91/03/1189/9 $2.00
The Journalof CellBiology,Volume 112,Number 6, March 1991 1189-1197
I 189
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sin, and myosin substrate turnover activity and cytoskeletal filament-driven translocation were examined
using 15 ATP analogues. The dyneins were more selective in their substrate utilization than bovine brain
kinesin or muscle heavy meromyosin, and even different types of dyneins, such as 14S and 22S dynein
from Tetrahymena cilia and the/~-heavy chain-containing particle from the outer-ann dynein of sea urchin.
flagella, could be distinguished by their substrate
specificities. Although bovine brain kinesin and muscle heavy meromyosin both exhibited broad substrate
specificities, kinesin-induced microtubule translocation
varied over a 50-fold range in speed among the various substrates, whereas heavy meromyosin-induced actin translocation varied only by fourfold. With both
Published March 15, 1991
firmed this in a quantitative manner, determining the apparent Kms and Vm~Sof turnover for naturally occurring NTPs
with 22S dynein from Tetrahymena cilia (Shimizu, 1987).
For ATP, this dynein has an apparent Km in the micromolar
range, while the apparent Km is two orders or more higher
for other NTPs. Kinesin is shown to have broad substrate
specificity with naturally occurring NTPs (Kuznetsov and
Gelfand, 1986; Porter et al., 1987; Cohn et al., 1989).
In the last few years, a cytoplasmic form of dynein
(Paschal and Vallee, 1987; Schroer et al., 1989), numerous
myosin-like motors (Kiehart, 1990) and several kinesin-like
motors (Vale and Goldstein, 1990) have been identified. One
of the challenges of the next few years is to identify the biological functions of these various motors. In some instances,
the phenotype of a cell with a mutation in the gene encoding
a motor protein provides some clue as to the motors. In some
instances, the phenotype of a cell with a mutation in the gene
encoding a motor protein provides some clue as to the motor's physiological role. Another approach, which is also
amenable in cells which do not have well-developed
genetics, is to reconstitute a motility event in vitro and probe
the system biocbemically. Previously, the ATPase inhibitors
vanadate and EHNA and an alkylating reagent NEM have
been used as the probes to identify what type of motor may
Glossary
ATPcxS
ATPflS
ATP3,S
didcoxy ATP
dimethyl ATP
8-azido ATP
8-bromo ATP
EHNA
etheno ATP
FTP
HMM
MAP
monomethyl ATP
NTP
PRTP
2~ATP
3~IATP
adenosine 5'-O-(bthiotriphosphate)
adenosine 5'-O-(2-thiotriphosphate)
adenosine 5'-O-(3-thiotriphosphate)
2' Y-dideoxy-adenosine 5"triphosphate
A~,N~-dimethyl-adenosine 5'-triphosphate
8-azido-adenosine 5'-triphosphate
8-bromo-adenosine 5'-triphosphate
erythro-9-[3-(2-hydroxynonyl)]adenine
1,N~-etheno-adenosine 5' triphosphate
formycin 5"triphosphate
heavy meromyosin
microtubule-associated protein
N~-methyl-adenosine 5'-triphosphate
nucleoside 5"triphosphate
purine riboside triphosphate
2'-deoxy-adenosine 5"triphosphate
3'-deoxy-adenoside 5'-triphosphate
Materials and Methods
Kinesin was purified according to a method developed by E Maiik and
R. Vale (manuscript in preparation). Briefly, a bovine brain high speed supernatant was warmed in the presence of 33% glycerol and 1 raM GTP
to polymerize microtubules. Then apyrase, to deplete nucleoside triphosphates, and 5'-adenylylimidodiphosphate (AMPPNP) (20 ~,M) were
added to the suspension to induce binding of kinesin to microtubules.
Microtubules pelleted by centrifugation were incubated with 5 mM Mg-ATP
to release kinesin. Kinesin was purified by pbosphocellulose (Whatman,
Kent, England) and Biogel A5M (Bio-Rad Laboratories, Richmond, CA)
gel filtration chromatography. As a last step, the enzyme was adsorbed onto
a 2 ml S-Sepharose (Pharmacia, Uppsaia, Sweden) column and then eluted
with 80 mM KCI. Fractions containing kinesin were pooled and then frozen
in liquid nitrogen in the presence of 10% sucrose; storage was at -80"C.
Microtubules were obtained from bovine or porcine brains by cycles of
polymerization and depolymerization, and MAP-free tubulin was prepared
by phosphocellulose (for translocation assay) or DEAE-Sephacel (for substrate turnover assay) column chromatography. Polymerization of the MAPfree microtubules was induced by 7% dimethylsulfoxide (Vale and Toyoshima, 1989a). In some cases, especially where the tubulin concentration
was low, an ~uimolar concentration of taxol was added to further stabilize
the microtubules. No detectable differences were observed with those microtubules in both assays irrespective of the starting materials or of the preparation methods.
Heavy meromyosin and actin were prepared from rabbit back muscles
by the methods of Okamoto and Seldne (1985) and of Spudich and Watts
(1971), respectively.
The protein concentrations of dyneins, actin, heavy meromyosin, and
tubulin were determined by the method of Lowry et ai. (1951), using bovine
serum albumin as a standard. The concentration of kinesin was estimated
by the method of Hackney (1988), where the densitometry of the kinesin
heavy chain band on an SDS-polyacrylamide gel was performed using bovine serum albumin as a standard.
ATP Analogues
For the chemical structure of ATP analogues used herein, see Fig. 1. ATP,
2'dATE dideoxy ATP, UTP, CTP, GTP, 8-bromo ATE and etheno ATP
were purchased from Sigma Chemical Co. (St. Louis, MO), Boehringer
Mannheim GmbH (Mannheim, Germany), or Pharmacia (Uppsaia,
Sweden) and purified by a DEAE-Sephadex A-25 column chromatography
with triethylammonium bicarbonate buffer Loll 7.6) gradient (from 0.1 to
0.6 M). 3~ATP and FTP were made from the corresponding monophosphate forms (Sigma Chemical Co.) with a trace amount of triphosphate form
by the adenylate ldnase and pyruvate ldnase (both enzymes from Boehringer
Mannheim GmbH) reaction and purified as above. To get rid of contaminating ATP (ca. 0.4%) from 8-azido ATP (Sigma Chemical Co.), it was treated
with 22S dynein until about half of 8-azido ATP was converted to the
diphosphate form, when most of contaminating ATP must have been turned
over because of highly preferred specificity of this enzyme to ATP. Then
the mixture was chromatographed as above and the triphosphate form was
obtained. PRTP, monomethyl ATP, and dimethyl ATP were synthesized
from the corresponding nucleosides (Sigma Chemical Co.) by two-step
chemical phosphorylation to the diphosphate forms (Shimizu and Furnsawa, 1986) and then by enzymatic phosphorylation to the triphosphate
forms by pyruvate ldnase with phospho¢nolpyrnvate. The final products
were also purified as above. The preparation method of the phosphorothioate analogues of ATP was described previously (Eckstein and Goody, 1976;
Shimizu et al., 1990). The purification of ITP to separate contaminating
ATP was carried out with a Dowex I column as described (Shimizu, 1987).
The purity of the nucleotides was checked by high performance liquid
chromatography with a Cls reverse phase column. All nucleotides were
substantially free from contamination, except for 8-azido ATP and
ATPflS(Sp). 8-azido ATP purified as above still contained ATP-like material, but not more than 0.1%. ATPflS(Sp) contained about 10% of the (Rp)
isomer.
Substrate TurnoverAssay
22S and 14S dyneins were obtained from cilia of Tetrahymena thermophila
SB-255 by the method of Porter and Johnson (1983). The B-heavy
chain-containing particle (fl-particle) was prepared from the 21S outer arm
The turnover rate of ATP or an analogue was determined from the time
course of the enzyme reaction. First, a rough estimate of the turnover rate
was obtained and then the assay was repeated under a suitable condition to
get a better value for the rate. The assay mixture contained: 50 mM MOPSNaOH (pH 7.0), 4 mM MgCI2, 1 mM ATP or analogue for dynein; 80 mM
imidazole-HCl (pH 6.g), 2 mM MgCI2, 1 mM EGTA, and 1 mM ATP or
The Journal of Cell Biology, Volume 112, 1991
1190
Proteins
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be powering a motile event. Dyneins, for example, are generally thought to be more sensitive to vanadate, EHNA and
NEM than kinesins. However, such generalizations using a
very limited number of perturbing agents could lead to erroneous interpretations (see Walker et al., 1990).
In the present study, we examined a different strategy for
distinguishing among different motor proteins. When different ATP analogues were tested for their ability to be turned
over or support in vitro motility by dynein, kinesin and heavy
rneromyosin motors, we found that the substrate utilization
by each motor tested was unique. Nucleotide "fingerprints"
may therefore be a useful diagnostic means of identifying
which type of motor is involved in a form of cellular motility
that can be reconstituted in vitro. One such application is described in the accompanying paper (Scbliwa et al., 1991).
dynein of sperm flagella from Japanese sea urchin, Pseudocentrotus
depressus, according to the method of Tang et al. (1982).
Published March 15, 1991
A
N.H2
N
N
H
H
adenine
pumm
NHCH3
N(CH3)2
N
N
H
I
N
H
_N6-rnethyladenine
N6,N_6-d'rnethyl
adenine
NH2
NH2
L..:..:8-brorno-aden~'~
8-azido-adenine
//-",,
H
(base of) forrnycin
B
0
_
H
1~6-etheno adenine
0
S
I
I
O-P.O-P-O-P-OAdenosine
0 0 0
ATI'BS
0 S 0
I
t
I
O.P.O-P-O-P-O-Adenos~ne
I
I
I
0 0 0
ATPrS
s 0 0
i
I
I
O-P.O-P-O-P-OAdenosine
I
I
I
0
0 0
Figure I. Chemical structures o f ATP analogues. (.4) Structure o f
modified adenines used in this study. (B) Structure o f the phosphorothioate analogues of ATP. Because o f the tetrahedral nature
o f phosphorus atom, two stereoisomers exist for ATPc~S and for
ATPBS, i.e., (Sp) and (Rp) isomers. They are referred to as
ATPc~S(Sp) and so on. Negative charge symbols and double bonds
are omitted to make the formula simpler and because of equivalence
o f all four b o n d s o f a phosphorus atom.
Turbidimetric Assay for the Dissociation of the
Microtubule-22S Dynein Complex
A suspension of microtubule (0.1 mg/ml)-22S dynein (0.18 mg/ml) complexes in 50 mM MOPS-NaOH (pH 7.0) and 4 mM MgCI2 maintained at
28"C was transferred into a cuvette and a small amount ofa nucleotide solution was added. The time course of the decrease in absorbance (turbidity)
at 420 nm due to nucleotide-induced dissociation was recorded for up to
4 min. The first time point available with this method was •5 s.
Detergent Models of Tetrahymena
The Tetrahymena models were made according to the method of Goodenough (1983) using NP-40 as a detergent with a slight modification
(Shimizu et al., 1990). The motion of reactivated cilia was recorded on
videotapes and the beat frequency was measured with slow speed playing.
For 1 determination, 20 cilia were analyzed.
Results
Substrate Specificities of Enzymatic and Motility
Activities of Dynein
Substrate TurnoverActivities of 22S and 14S Dyneinsfrom
Tetrahymena Cilia. The enzymatic activity of 22S dynein toward the ATP analogues (Fig. 1 for the structures) was inves-
The in vitro microtubule translocation assay was carried out as described
tigated using Lineweaver-Burk plots (Table I). The V~ and
apparent Km of turnover of ATE 2'dATP, or 3'dATP were
comparable, whereas removal of both 2' and 3' oxygens
(dideoxy ATP) increased the apparent Kmfrom 2 to 40 #M.
All three of these deoxy derivatives gave simple MichaelisMenten type relationships.
Monomethyl ATP was the only ATP analogue giving complex kinetics amongst the substrates investigated; the Lineweaver-Burk plot gave a downward bend as in the case of
ATP and ATP'yS (Shimizu et al., 1989), with the apparent
Km and V~ being comparable to those of ATP turnover.
FTP, one of the two fluorescent ATP analogues investigated
herein, was also a good substrate. The V~, of 8-bromo
ATP was even higher than that of ATP, although the apparent
Km was high (30/~M).
Dimethyl ATP, PRTP, etheno ATP (another fluorescent
ATP analogue), and 8-azido ATP (often used as a probe for
the adenine-binding sites of certain enzymes) were all turned
over poorly by 22S dynein.
The other form of dynein from Tetrahymenacilia is 14S
dynein. The catalytic activity of 14S dynein toward the ATP
analogues was measured at 1 mM substrate as shown in Ta-
Shimizu et al. Nucleotide Specificityof Molecular Motors
1191
an analogue for kinesin; 25 mM Hepes-NaOH (pH 7.4), 25 mM KCI, 4 mM
MgC12, 1 mM ATP or an analogue for HMM. The enzyme concentration
in the assay solution was 5-25/~g/ml. After incubation at 25°C, the reaction
was terminated by the addition of final 0.3 M perchloric acid. Precipitates,
if formed, were removed by centrifugation and the supernatant was assayed
for phosphate by the modified malachite green method (Shimizu and
Furusawa, 1986).
When measuring the microtubute-activation of kinesin or actinactivation of HMM, the polymerized microtubules or actin filaments were
diluted with a nucleotide-free buffer solution, pelleted by centrifugation,
and resuspended in the same nucleotide-free solution containing trace
amounts of taxol or phalloidin, respectively, in order to reduce the background of GTP or ATP contained in the protein solution. By repeating this
treatment once more, the contribution of GTP or ATP from the protein solution to the assay solution was significantly <1 tLM.
For determining the Michaelis constant of 22S dynain for ATE 2'dATP,
or YdATP, the coupled assay method using pyruvate kinase and phosphoenolpyruvate was used, and the production of corresponding diphosphate was measured to determine the substrate turnover rate of dynein
(Shimizu, 1981).
In Vitro Translocation Assay
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ATP~S
'
(Vale and Toyoshima, 1989b) using a flow chamber method. A dynein or
kinesin (50 #g/ml) solution was introduced into a flow chamber consisting
of a slide glass and a eoverslip separated by spacers; after washing with a
buffer solution, a mixture of taxol-stabilized microtubules (20 #g/ml) and
ATP or an analogue (1 raM) was then perfused.The buffer solution ingredients were: 10 mM Tris-acetate (pH 7.5), 50 mM potassium acetate, 4 mM
MgSO4, 1 mM EGTA, and 1 mM DTT for 14S and 22S dynein; 10 mM
Tris-HCl (pH Z5), 50 mM KCl, 4 mM MgCIe, 1 mM EGTA, and 1 mM
DTT for/~-partiele; 80 mM Pipes-NaOH (pH 6.8), 2 mM MgCI2, and
1 mM EGTA for kinesin. The translocation of microtubules was observed
with a darldield microscope and recorded on a video tape. The speed of
translocation was analyzed by replaying the videotapes afterwards. 20 microtubules were analyzed for one substrate to get the average and standard
deviation.
In the case of aetin translocation assay, an HMM (50 t~g/ml) solution was
applied to a flow chamber covered with a nitrocellulose film and a solution
of actin (0.5/~g/ml) labeled with rhodamine-phalloidin was perfused as described by Toyoshima et al. (1987). The movement of actin upon addition
of ATP or an analogue (1 mM) was observed with a fluorescence microscope and analyzed as above. The buffer solution consisted of 25 mM
imidazole-HC1 (pH 7.4), 25 mM KC1, 4 mM MgC12, and 1 mM DTT.
Published March 15, 1991
Table L Nucleotide Specificity of Microtubule-22S Dynein System
Substrate turnover
Microtubule transloeation
Nucleotide
ATP
+ Triton (0.1%)
V~,
Apparent Km
izmls
l~mls
,umollmin/mg
/zM
3'dATP
Dideoxy ATP
4.52
1.26
2.00
0.94
Monomethyl ATP
Dimethyl ATP
PRTP
0.28 ± 0.075 (6.2)
-
2'dATP
±
±
±
±
- Triton
1.06
0.31
0.52
0.31
(100%)
(28)
(44)
(21)
11.2 ± 1.38 (248%)
N.M.
N.M.
4.80 ± 0.85 (106)
0.25, 0.30
0.31
0.51
0.56
0.69, 2.0
3.5
6.3
41
0.90 ± 0.19 (20)
-
0.063, 0.19
0.016
0.13
1.0, 7.1
83
96
8-Bromo ATP
8-Azido ATP
-
-
0.55
0.044
29
170
FTP
Etheno ATP
-
0.054 ± 0.017 (1.2)
-
0.29
0.0088
10
210
1.34"
0.82*
0.087*
0.007"
0.052, 0.11*
8.2*
83*
67*
105"
<1, 20*
ATPotS(Sp)
ATPotS(Rp)
ATP/~S(Sp)
ATPBS(Rp)
ATP3~S
0.83 ± 0.24 (18)
0.21 ± 0.11 (4.6)
1.86 ± 0.45 (41)
0.26 + 0.19 (5.8)
ble II. The relative turnover rates to that of ATP were similar
to those of 22S dynein except for 8 substituted analogues
whose relative turnover rates were somewhat different between the two dyneins.
In Vitro Microtubule Translocation by 22S Dynein, 14S
Dynein, and the B-Parficlefrom Sea Urchin Sperm FlagellarOuterArm Dynein. Each analogue (at 1 mM concentration) was examined for its ability to support in vitro microtu-
Table II. Nucleotide Specificity of Microtubule-14S Dynein and Microtubule-13-Particle System
14S dynein
Nucleotide
Substrate turnover rate
/3-Particle
Microtubule translocation
/~mollmin/mg
microtubule translocation
izm/s
+
+
+
+
0.70
0.51
0.36
0.48
itm/s
ATP
2'dATP
3'dATP
Dideoxy ATP
0.112
0.112
0.138
0.158
4.29
4.24
3.30
2.86
(100%)
(99)
(77)
(67)
Monomethyl ATP
Dimethyl ATP
PRTP
0.0665
0.0127
0.0359
0.73 + 0.098 (17)
-
8-Bromo ATP
8-Azido ATP
0.0908
0.0676
1.17 + 0.24 (27)
=
3.42 + 0.53 (35)
FTP
Etheno ATP
0.0845
0.0042
0.084 + 0.019 (2.0)
-
4.16 + 0.52 (43)
=
ATPc~S(Sp)
ATPtxS(Rp)
ATP~S(Sp)
ATP/SS(Rp)
ATP~S
0.157
0.141
0.0040
0.0013
0.0843
1.20 ± 0.13 (28)
0.52 ± 0.24 (12)
8.43 ± 1.19 (87)
=
=
=
0.51 ± 0.17 (5.3)
-
9.65
8.85
11.9
5.28
+
+
+
+
1.20
1.11
1.03
0.76
(100%)
(92)
(123)
(55)
3.52 + 0.38 (36)
=
=
The substrate turnover rate of 14S dynein toward each nucleotide (1 raM) was assayed at 250C in the same manner as that of 22S dynein as described in Materials
and Methods. The remover rate is expessed as ~,mol/min/mg 14S dynein. The microtubule translocation assay at 1 mM nucleotide was also performed in the same
manner. = indicates that the microtubules did not associate with the E-particle-coated glass surface but were floating in the solution. The relative translocation
speed is also shown in the parentheses with the speed with ATP as 100%.
The Journal of Cell Biology, Volume 112, 1991
1192
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The substrate turnover activity and the microtubule transloeation speed were measured at 250C as described in Materials and Methods. The V~, and apparent
Km for each substrate were estimated from the Lineweaver-Burk plot. This dynein exhibits a downward bend in this plot with ATP (Shimizu, 1981), ATPTS
(Shimizu et al., 1989) or with monomethyl ATP, so that two values are listed for these substrates. The values for the phosphorothioate analogues of ATP except
for ATP'yS are taken from the previous study with 22S dynein from Z pyriformis (Shimizu and Furnsawa, 1986), which are marked with an asterisk. Those for
ATP),S are from Shimizu et al. (1989) with 22S dynein from 7". thermophila assayed at 28"C, marked with a dagger. V~, is expressed in/~mol/min/mg 22S
dynein. + Triton indicates that the substrate solution for the translocation assay contained 0.1% Triton X-100. The relative translocation speed is also shown
in the parentheses with the speed with ATP in the absence of Triton X-100 as 100%. N.M., not measured.
Published March 15, 1991
Table IlL Reactivation of Ciliary Motility
Beat frequency (s -~)
at the substrate concentration of
Nucleotide
ATP
2'dATP
YdATP
dideoxy A T P
PRTP
Dimethyl-ATP
Monomethyl-ATP
1 mM
12.4
12.6
12.7
9.0
5555-
1.0
1.4
0.8
0.7
2.9 5- 0.2
0.3 mM
0.1 mM
4.5 + 0.5
8.5 + 1.1
7.2 5- 1.1
3.5 + 0.3
-
0.62 + 0.08
8-Bromo A T P
8-Azido A T P
FTP
Etheno A T P
The preparation of the NP-40 models of T. thermophila and the analysis of the
ciliary reactivation were performed as described in Materials and Methods.
Dideoxy ATP or monomethyl ATP did not support the ciliary motility at 0.1
mM but at 0.3 raM, the reactivation was observed and the beat frequency is
listed. Others were not checked at 0.3 raM. - , no reactivation observed. As
for the phosphorothioate analogues, only ATPctS(Sp) was shown to be able to
support the motility (Shimizu et al., 1990); beat frequency was 4.6 + 0.6 s-~
at 1 raM.
Table IV. Nucleotide Specificity of Dissociation of the Microtubule-22S Dynein Complex
Dissociation at the nucleotide concentration of
Nucleotide
0.25 raM
1 mM
ATP
2'dATP
YdATP
Dideoxy A T P
Rapid
Rapid
Rapid
Rapid
Monomethyl A T P
Dimethyl A T P
PRTP
Rapid
None
Slow, incomplete
Slight
Slow (faster), complete
8-Bromo A T P
8-Azido A T P
Slow, incomplete
None
Slow (faster), almost complete
Slight
FTP
Etheno A T P
Rapid
None
None
ATPtxS(Sp)
ATPt~S(Rp)
ATP/3S(Sp)
ATP/3S(Rp)
ATP'yS
Rapid
Slow, incomplete
None
None
Rapid
G T P (0.5 raM)
ITP (0.5 raM)
UTP (0.5 raM)
CTP (0.5 raM)
Slow, incomplete
None
Slight
None
Slow, almost complete
The dissociation of the microtubule-22S dynein complex was measured by monitoring the turbidity of the suspension at 420 nm for up to 4 min after addition
of ATP or analogue as described in Materials and Methods. Terms to describe the dissociation are defined as follows: the extent of dissociation is complete (saturating amount of ATP, e.g., 0.2 mM, would not cause further turbidity drop) or not complete (addition of ATP did cause further turbidity drop), and the latter is
subdivided into: almost complete (>80% completion of turbidity change), incomplete (<80%), slight (<20%), or none (<5 %). The extent of incomplete dissociation
varies considerably depending on the substrate concentration. As for the rate of dissociation, it is rapid (complete within 5 s) or slow (not complete within 5 s).
Shimizu et al. Nucleotide Specificity of Molecular Motors
1193
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bule translocation on 22S dynein-coated glass surface (Table
I). All the deoxy ATP derivatives supported the translocation
of the microtubules, although the speed was slower. Among
adenine-modified analogues, only monomethyl ATP was
competent for supporting the movement. Of the phos-
phorothioate analogues, ATPotS(Sp), which was a good substrate for dynein catalytic activity (Shimizu and Furusawa,
1986), supported the microtubule translocation. Vale and
Toyoshima (1989a) reported that ATP3,S could induce slow
but significant microtubule translocation mediated by 22S
dynein, and we confirmed this with column-purified, substantially ATP-free ATP3,S at concentrations between 0.3
and 2 raM.
As Vale and Toyoshima (1989a) demonstrated, addition of
0.1% Triton X-100 improved the 22S dynein-mediated
microtubule translocation by increasing the speed, making
movement more continuous and making more microtubules
move (Table I). In addition, slow movement was observed
with FTP in the presence of Triton X-100, but other NTPs
were not competent even in its presence.
Next the microtubule translocation assay at 1 mM substrate on the 14S dynein-adsorbed glass surface was performed (Table 1I). The results were also similar to those of
22S dynein, although the specificity was somewhat broader.
In addition to those analogues that supported the 22S dyneinmediated microtubule translocation, FTP and, surprisingly,
8-azido ATP supported the motility. We believe that 8-azido
ATP itself, and not contaminating ATP-like material in 8-azido ATP, was responsible for the movement. The maximum
of ATP contamination in our 8-azido ATP was 0.1%, but motility was observed at 0.1 mM 8-azido ATP (contamination
of ATP was 0.1 #M at most) at about half the speed of that
at 1 mM. ATP~S supported the microtubule translocation as
in the case of 22S dynein as above, although more slowly.
Dialysis of the outer arm 21S dynein from sea urchin
Published March 15, 1991
Table V. NucleotideSpecificityof Microtubule-Kinesin System
Substrate turnover rate
Nucleotide
Microtubule translocation
ATP
2'dATP
3'dATP
Dideoxy A T P
0.422
0.382
0.304
0.290
- microtubules
,umls
,umollmirdmg kinesin
0.0056
0.0046
0.0125
N.M.
0.572
0.532
0.353
N.M.
0.198 + 0.052 (47)
0.086 5 : 0 . 0 1 8 (20)
0.0075 5 : 0 . 0 0 5 1 (1.8)
0.0061
0.0169
0.0335
0.603
0.449
0.284
8-Bromo A T P
8-Azido A T P
0.013 5 : 0 . 0 0 6 7 (3.1)
-
0.0151
0.0245
0.251
0.196
FTP
Etheno A T P
0.037 + 0.019 (8.8)
0.054 5 : 0 . 0 2 8 (13)
0.0210
0.0235
0.427
0.377
ATP~xS(Sp)
ATPc~S(Rp)
ATP~S(Sp)
ATP/~S(Rp)
ATPTS
0.077 5 : 0 . 0 2 3 (18)
"0.0091 5 : 0 . 0 0 2 7 (2.2)
0.0094
0.0451
0.0113
N.D.
N.M.
0.520
0.194
0.303
N.D.
N.M.
Monomethyl A T P
Dimethyl A T P
PRTP
5:0.062
5:0.074
5:0.059
5:0.049
+ microtubules
(100%)
(91)
(72)
(69)
The substrate turnover assay and the microtubule translocation assay were performed at 25 and 22-24°C, respectively, as described in Materials and Methods.
The substrate turnover by kinesin (10 #g/ml) was measured in the absence or the presence of the MAP-free microtubules (0.95 mg/ml). The concentration of
nucleotide was 1 mM for both types of assay. The relative translocation speed is shown in the parentheses with the speed with ATP as 100%. N.D. and N.M.,
n o t detectable and not measured, respectively.
Reactivation of Ciliary Motility of Detergent Models of
Tetrahymena. NP-40 mdels of Tetrahymenacilia were reactivated only by ATE deoxy ATP derivatives and monomethyl
ATP (Table III). Neither FTP, 8-azido ATE nor ATPTS
(at 1 mM) supported ciliary reactivation. Each deoxy ATP
derivative at 1 mM produced a beat frequency similar to that
evoked by ATP, whereas the frequency was smaller at 0.1 mM
than that at 0.1 mM ATP. Dideoxy ATP at 0.1 mM did not
support motility. Monomethyl ATP induced slower ciliary
beating and, as dideoxy ATE it did not support the motility
at 0.1 mM.
Turbidimetric Measurements of Dissociation of the Microtubule-22SDyneinComplex.We also examined the ability of the various ATP analogues to induce the dissociation
of the microtubule-22S dynein complex (Porter and Johnson, 1983; Shimizu and Furusawa, 1986). As shown in Table
The Journal of Cell Biology, Volume 112, 1991
IV, each of the deoxy ATP derivatives, monomethyl ATE
and FTP (0.25 mM) induced rapid dissociation (complete
within 5 s). ATPaS or ATPTS were previously demonstrated
to rapidly dissociate the complex (Shimizu and Furusawa,
1986), confirmed herein. Of the other analogues that did not
induce rapid dissociation, only PRTP and 8-bromo ATP induced nearly complete, but slow dissociation at 1 mM concentration. Naturally occurring NTPs at 0.5 mM could not
substitute for ATP except CTP which induced slow but complete dissociation within 2 min.
Nucleotide Specificity of the
Microtubule-Kinesin System
Kinesin-induced motility of the microtubules was reported
to exhibit a rather broad substrate specificity; each of naturally occurring NTPs at 10 mM could support motility of
microtubules along kinesin-coated surfaces, though the
translocation speed varied from one substrate to another
(Porter et al., 1987; Cohn et al., 1989).
As shown in Table V, most ATP analogues supported
kinesin-induced microtubule movement while only 8-azido
ATE ATPo~S(Rp), or ATP/~S isomers were incompetent for
inducing the motility. The speed of microtubule translocation induced by the ATP analogues, however, varied to a considerable extent. ATP itself was the best substrate in terms
of the speed of translocation and the deoxy derivatives were
next. Those with modified adenine moieties produced slow
translocation. ATPTS or PRTP (1 mM) induced movement
at 2 % the speed of 1 mM ATP.
The substrate turnover activity of kinesin in the absence
or presence of 0.95 mg/ml microtubules also varied depending on the substrate. The turnover was accelerated by
microtubules in all cases tested (Table V) including naturally
occurring NTPs (data not shown). With ATP, the activation
1194
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sperm flagella against a low ionic strength solution dissociates the two-headed dynein into single-headed o~- and B-particles (Tang et al., 1982). While the a-particle does not mediate microtubule translocation, the 13-particle adsorbed on
glass coverslip is able to induce motility (Sale and Fox,
1988; Vale et al., 1989). As shown in Table II, the nucleotide
specificity of microtubule translocation on B-particle-coated
glass surfaces was similar to that of 14S dynein-mediated
movement, except that ATP,',S(Sp) and FTP supported better
microtubule translocation on fl-particle-coated glass surface
than on 14S dynein-coated surface. Previously, it was noted
that microtubules did not adhere to glass surfaces coated
with/3-particles in the absence of ATP (Sale and Fox, 1988;
Vale et al., 1989) in contrast to the results with 14S or 22S
dynein. With several ATP analogues, microtubules did not
adhere to the H-particle-coated surface, which may indicate
a weak interaction between the ~-particle and those ATP
analogues.
Published March 15, 1991
Table VI. Nucleotide Specificity of Actin-HMM System
Substrate turnover rate
Nucleotide
Actin translocation
- actin
i~m/s
ATP
2'dATP
3'dATP
Dideoxy A T P
5.21
4.51
4.62
5.43
+
+
+
+
Monomethyl A T P
Dimethyl A T P
PRTP
3.80 + 0.53 (73)
2.96 + 0.39 (57)
2.38 + 0.32 (46)
8-Bromo A T P
8-Azido A T P
1.04
0.87
0.53
0.79
+ actin
izmol/min/mg HMM
(100%)
(87)
(89)
(104)
-
0.0105
0.0141
0.0200
0.0300
0,257
0,343
0.626
0,352
0.0454
0.0528
0.0547
0.442
0.677
0,477
0.215
0.188
0.412
0.420
FTP
Etheno A T P
1 . 3 2 : 1 : 0 . 2 6 (25)
1.18 5 : 0 . 3 4 (23)
0.0144
0.0930
0.422
0.584
ATPtxS(Sp)
ATPo~S(Rp)
ATP/3S(Sp)
ATP/~S(Rp)
ATP3,S
10.4 + 1.01 (200)
0.52 + 0.16 (10)
-
0.0311
0.357
0.137
0.00082
N.M.
0.707
0.630
0.0470
<0.0005
N.M.
The substrate turnover assay and the actin translocation assay were performed at 25"C as described in Materials and Methods. The concentration of nucleotide
was 1 mM for both types of assay, The substrate turnover by HMM (10 t~g/ml) was measured in the absence or the presence of F-actin (0.44 mg/ml). The relative
transloeation speed is shown in the parentheses with the speed with ATP as 100%. N.M., not measured.
The substrate turnover rate of HMM was, in most cases,
higher in the presence of actin than in its absence (Table VI),
but again, the magnitude of activation or the turnover rate
in the presence of actin does not seem to correlate with the
speed of actin translocation. It should be noted that even with
8-substituted ATP analogues, which did not support the motility, the activation of substrate turnover by actin was observed, as in the case of the microtubule-kinesin system.
Nucleotide Specificity of the Actin-HMM System
The catalytic activity of myosin and actin-myosin-based motility systems have long been known to exhibit broad substrate specificity (Tonomura et al., 1967; Takenaka et al.,
1978). The in vitro translocation system with actin and
HMM is shown to be supported by CTP or UTP (Toyoshima,
Y., S. Kron, J. Spudich, and H. White, manuscript in preparation).
Our experiments also demonstrated that many of the ATP
analogues supported the HMM-mediated actin translocation
in vitro (Table VI). Of the analogues examined, only 8substituted ATP analogues, ATPBS isomers and ATP~S
were incompetent for supporting motility. In this regard, the
actin-HMM system was similar to the microtubule-kinesin
system. A notable difference, however, was that while the
speed of translocation varied significantly in the microtubnlekinesin system depending on the substrate species, most ATP
analogues induced translocation at 1-5 •rrds in actin-HMM
system. Two exceptions were the ATPotS isomers; ATPc~S
(Rp) supported actin movement at 10 % of the speed of that
by ATP, and ATPctS(Sp) induced actin translocation at twice
the rate of ATE This latter result is the only case where an
ATP analogue induced filament movement at a significantly
faster rate than ATP with the three in vitro translocation systems studied herein.
Shimizu et al. Nucleotide Specificity of Molecular Motors
Discussion
Nucleotide Specificities of Dynein, Kinesin, and HMM
Previously, we have shown in a quantitative manner that the
catalytic activity of 22S dynein is highly specific for ATP
(Shimizu, 1987). Here, we extended the survey to include
a wider variety of ATP analogues and several other molecular motors such as 14S dynein, HMM, and bovine brain
kinesin. Myosin, or its proteolytic subfragment conveying
the enzymatic sites such as HMM, has long been known to
have a broad substrate specificity. This was confirmed by the
present results that all the adenine-modified analogues and
deoxy-derivatives of ATP examined were turned over faster
than ATP itself. Kinesin also exhibited broad substrate
specificity; ATP again was turned over slower than many analogues. Thus, these two molecular motors are similar to
each other in this context, but different from dyneins.
The specificity of 22S dynein substrate turnover was investigated in a quantitative manner. Slight modification of the
ribose moiety of ATP did not seem to affect the 22S dyneinsubstrate interaction (also see Inaba et al., 1989). On the
other hand, modification of the adenine moiety of ATP
resulted in a serious reduction in its capability to act as a substrate for 22S dynein in most cases (see Omoto and
1195
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by microtubules was •100-fold. It is of interest to note that
neither the relative magnitudes of activation by the microtubules nor the absolute turnover rates in the presence of
microtubules corresponded to the relative speeds of the
microtubule translocation induced by the ATP analogues.
Even with the nucleotides that did not support the motility,
activation of the turnover rate was still observed in the presence of the microtubules.
Published March 15, 1991
Potential Applications of Using Nucleotides to
Identify Molecular Motors
Our results demonstrate that different motors, including several forms of dynein, exhibit distinct substrate specificities
in the in vitro motility assay. Useful criteria for comparing
the activities of different motors are (a) the ability or inability ofa substrate to support movement, and (b) for those substrates that induce translocation, the relative velocity compared to that induced by 1 mM ATP. These tests performed
with a battery of ATP analogues reveal a nucleotide "fingerprint" of the enzymatic activity of a motor that may be useful
in identifying what type of motor may be operating in a form
of cell motility. This strategy may be more convincing than
using the concentration dependence of vanadate or EHNA
inhibition or NEM susceptibility.
In the accompanying paper (Schliwa et al., 1991), this approach was applied to examine the motor that drives organelle transport in Reticulomyxa. Since it is now possible
to reconstitute mitotic anaphase B movements in vitro
(Cande and McDonald, 1985; Masuda et al., 1990), these
ATP analogues may be useful for gaining insight into what
motors are powering these processes. Kinetochore-based
movements and various forms of membrane transport that
can be reconstituted in vitro may similarly benefit from this
type of characterization.
To interpret substrate specificity of a form of cellular motility in terms of what motor may drive this process, one
must analyze more purified motors for their substrate utilization in the in vitro motility assay. In addition to the motors
described herein, cytoplasmic dynein (Collins and Vallee,
1989) and dynamin (Shpetner and Vallee, 1989), two
The Journal of Cell Biology, Volume 112, 1991
microtubule-based motors, should be examined. Furthermore, a variety of myosin-like and kinesin-like proteins have
been uncovered through genetic and molecular biological
techniques. Many of these motors can be expressed in bacteria and their motility is characterized (Walker et al., 1990).
It will be interesting to explore whether the utilization of
ATP analogues can distinguish between members of the
kinesin and myosin superfamilies.
The strategy of identifying what motor may drive a cellular process based on substrate specificity may have possible
problems as well. In the case of ciliary reactivation, the
specificity is more strict than microtubule translocation in
vitro: ATP3,S or FTP, which supported dynein-mediated
translocation, did not support ciliary reactivation. This may
mean that a motility system might exhibit different specificity
from that of reconstituted translocation system consisting of
the motor obtained thereof. On the other hand, some form
of motility, such as ciliary beating, may require the actions
of several motors, which may make interpreting the substrate
specificity difficult. The slow speed of translocation produced by some analogues in vitro may be also insufficient to
initiate ciliary beating in a reconstituted assay.
Implications for the Force-generatingCycle
The finding that ATP3,S supported kinesin- and dyneindriven microtubule translocation but not HMM-driven actin
movement is interesting in light of the enzymatic reaction
mechanisms of these molecular motors. With all three motors, product release is rate limiting and is ordered; phosphate is released first and ADP, second. According to kinetic
investigations, the phosphate release step, or the step immediately after phosphate release, is likely to be related to
power production in the actin-myosin system (Hibberd and
Trentham, 1986). ATP3,S may not support actin-HMM motility because the power production step does not function
well with thiophosphate, one of the products of ATP-~S hydrolysis. On the other hand, in the kinesin and dynein systems, power production is postulated to be related to ADP
release and not to phosphate (thiophosphate) release (Hackney, 1988; Holzbaur and Johnson, 1989). This could explain
why kinesin and dynein can use ATP~,S as an energy donor
for motility.
Filament-activated NTP turnover activity is generally
thought to be coupled to power production. In this study, we
found that actin or microtubules accelerated the turnover of
certain ATP analogues by HMM or kinesin, respectively,
even though these analogues did not support filament movement under similar experimental conditions. Such uncoupling may occur as the result of some steric difficulty or if
the step responsible for the accelerated substrate turnover
by either actin or microtubules is different from that involved
in the power production. Vallee and his group (Paschal and
Vallee, 1987; Shpetner et al., 1988) reported that CTP turnover by cytoplasmic dynein was much faster than that of ATP
but not accelerated by microtubules, and that the in vitro
microtubule translocation was not supported by CTP. Their
result is distinguished from the present result in that we observed filament-activated substrate turnover of certain ATP
analogues by kinesin or HMM without filament translocation.
It is of interest to note that nucleotides that supported
microtubule movement on 22S dynein-coated glass surfaces
also induced rapid dissociation of the microtubule-22S
1196
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Nakamaye, 1989). 14S dynein seemed to exhibit specificity
similar to 22S dynein (Table I).
The dynein, kinesin, and HMM translocation systems also
exhibited distinct differences in substrate specificity. While
the dynein-based systems showed quite narrow specificity,
the kinesin and HMM systems showed broad specificity. For
example, dimethyl ATP and etheno ATP supported fairly
good filament translocation with HMM and kinesin but not
with dynein. An intriguing difference between the kinesin
and HMM systems is found in the speed of translocation.
With the HMM system, variation in the speed of actin translocation was moderate and independent of the analogues,
while with kinesin system, the speed of the microtubule
translocation varied to a large extent. For instance, PRTP induced fairly fast translocation with the actin-HMM system
while the speed of kinesin-mediated microtubule translocation was only 2 % that of the ATP-induced one. In addition,
8-bromo ATP supported the kinesin system but not HMM
system.
Among dynein-mediated microtubule-translocation systems, an interesting difference was observed with 8-azido
ATP. This ATP analogue was a poor substrate for turnover
and translocation activity of 22S dynein but supported the
microtubule translocation mediated by 14S dynein or the
/~-particle. 8-azido ATP has been reported to be a good substrate for Chlamydomonas dyneins (Pfister et al., 1984,
1985) and for sea urchin flagellar or egg dynein (Pratt,
1986). It is reported not to be a good substrate for squid
dynein (Gilbert and Sloboda, 1989). Thus, 8-azido ATP may
be a useful analogue for distinguishing different types of
dynein molecules.
Published March 15, 1991
dynein complex, whereas those which did not induce rapid
dissociation did not support translocation. It seems likely
that the rapid dissociation of dynein heads from the microtubules is prerequisite to power production in the cross-bridge
cycle. If dissociation is slower than the hydrolytic step, a considerable fraction of hydrolysis and product release would
take place without dissociation, which would result in the
uncoupling of substrate turnover from power production, or
even a resistance to the motility. Thus, the ability of a nucleotide to dissociate the motor-filament complex may correlate with its ability to induce movement. We intend to examine whether this correlation between dissociation and
movement holds true for the kinesin and HMM systems
as well.
Shimizu et at. Nucleotide Specificity of Molecular Motors
1197
We would like to thank Ms. Hiroko Nishi for her secretarial assistance.
This study was supported by a grant-in-aid from Agency of Industrial
Science and Technology, MITI, to T. Shimizu.
Received for publication 30 July 1990 and in revised form 5 December
1990.
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